Sensitive – efficient amplification for reliable genotyping from even very limiting amounts of sample

Flexible – suitable for genotyping from a wide range of samples including human, animal and plant samples and compatible with many dual-labelled probe assays

Product Description

SensiFAST Hi-ROX Genotyping Kit has been developed for fast, precise and highly reproducible genotyping of sequence variants, including loci with type IV SNPs. The SensiFAST Hi-ROX Genotyping Kit is a combination of the latest advances in buffer chemistry, together with an antibody-mediated hot-start DNA polymerase system. This produces highly-specific, ultra-sensitive real-time PCR with clear allelic discrimination and outstanding allele clustering. The SensiFAST Hi-ROX Genotyping Kit is compatible with many dual-labelled probe assays, including TaqMan® probe based assays and has been validated on commonly used real-time instruments.

The advanced buffer chemistry in SensiFAST Hi-ROX Genotyping Kit provides higher stringency and more specific binding of the allele-specific probes, resulting in much narrower probe melting temperature windows. The combination of these developments leads to wider and clearer separation of allele clusters. The kit allows robust genotype calling, enabling reproducible and accurate results. The advanced buffer also means that minimal optimization is required to achieve fast, reproducible and accurate results, ideal for high-throughput in large-scale genotyping projects.

Applications

Single nucleotide polymorphisms (SNPs) types I, II, III and IV

Fig. 1 Discrete fluorescent signals for more accurate allele calls

DNA samples from ninety six patient were genotyped using a custom TaqMan drug metabolism genotyping assay, to detect an A/G SNP on gene CYP11B1. SensiFAST Genotyping Kit was used alongside a kit from supplier A, to illustrate the defined clusters with the SensiFAST Genotyping Kit, whilst supplier A gives poorly defined trailing clusters and low confidence calling.

Discrete fluorescent signals for more accurate allele calling.

Introduction to SensiFAST

Bioline’s SensiFAST Genotyping mix allows very sensitive discrimination between closely-related SNPs. In comparison with other kits on the market, the kit allowed clear discrimination between homo and heterozygotes, with clear resolution between the groups.

Polymerase activity during the reaction set-up causes non-specific amplification including primer-dimer formation. To avoid non-specific amplification the polymerase is only activated after heating at 95&degC for 2-3 minutes.

When comparing SensiFAST with a mix from another supplier we strongly recommend to use the suggested conditions for PCR setup and cycling. Every real-time mix has its specific composition and different properties. That’s why they should be tested with their specific conditions, mostly this is possible on different plates only. We recommend amplifying from a ten-fold template dilution series. Loss of detection at low template concentration is the only direct measurement of sensitivity. An early Ct value is not an indication of good sensitivity, but rather an indication of speed.

The recommended PCR conditions in the manual refer to the set-up of TaqMan probe-based PCR. If you want to use other probe types, it may be necessary to adapt the PCR setup conditions, please refer to the relevant literature. In case of hybridization probes it would be necessary to switch to a three-step PCR setup with annealing at 60°C and extension at 72°C and acquire the data at the end of the annealing phase (the exact annealing temperature may vary and has to be optimized for a specific assay). The two-step protocol with an annealing / extension step at 60°C would lead to the degradation of these probes, which is wanted for TaqMan but not for hybridization probes.
If you have further questions, please contact Bioline Technical Support.

The emission recorded from ROX during the baseline cycles is used to normalize the emission recorded from the reporter (SYBR®) in later cycles in some instruments. ROX compensates for small fluorescent fluctuations such as bubbles and well-to-well variations that may occur.

This method is only effective if all the researchers in the laboratory, or using the thermocycler, are all using a dUTP/dNTP and UNG system. If even one researcher is not, it ceases to be an effective control. Using dUTP has also been shown to be inefficient as it increases the Ct values by reducing reaction efficiency. Most labs do not need to use the dUTP method of control, optimised protocols will allow high specificity of PCR and good lab practices (using disposable consumables, the use of filter tips and maintaining a separate area for PCR set-up and PCR amplification and any post-PCR analysis) virtually eliminate the risk of cross contamination.